The endocrine role of the kidney as the producer of the potent calcigenic steroid hormone, 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) is now well recognized. The renal production of 1,25-(OH)2-D3 is stringently regulated according to the calcium demand of the organism. Although a number of possible regulators of this metabolic step have been proposed, the degree to which they affect renal 1,25-(OH)2-D3 production directly and the molecular mechanisms of their actions remain unknown. Thus the primary objective of the proposed research is to use the primary culture of chick kidney cells as an experimental system to determine which of the regulators of 25-OH-D3 metabolism have direct effects on renal cells and to examine the mechanism of their action in this system. Secondly, this culture system will be utilized to investigate the production of vitamin D-dependent calcium binding protein and as a bioassay for avian parathyroid hormone (PTH). The latter application will allow the examination of the regulation of PTH levels in the chick and the relationship between PTH, vitamin D status and the renal metabolism of 25-OH-D3 in vivo. Compounds to be tested for their effects on 25-OH-D3 metabolism by chick kidney cells in culture include PTH, calcitonin, prolactin, somatomedin, vitamin D metabolites, estrogens, glucocorticoids, and extra- and intracellular calcium and phosphorous levels. Compounds which affect 25-OH-D3 metabolism will be tested for the time course of their action, their effect on cyclic nucleotides, and their dependence on RNA and/or protein synthesis. The possible interaction of two or more modulators of 25-OH-D3 metabolism will also be examined. It is anticipated that results from these studies will provide a clear insight into the modes of interaction and interrelationships of the important regulators of calcium homeostasis.